RNA polymerase and transcription elongation factor Spt4/5 complex structure

Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases.

[1]  M. Gottesman,et al.  Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro , 1995, Journal of bacteriology.

[2]  K. Yano,et al.  DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. , 1998, Genes & development.

[3]  S. Darst,et al.  A Structural Model of Transcription Elongation , 2000 .

[4]  P. Cramer,et al.  Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution , 2001, Science.

[5]  R. Huber,et al.  Crystal structures of transcription factor NusG in light of its nucleic acid‐ and protein‐binding activities , 2002, The EMBO journal.

[6]  D. Court,et al.  A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants. , 2003, Biochemistry.

[7]  H. Handa,et al.  NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. , 2003, Genes & development.

[8]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[9]  R. Landick.  The regulatory roles and mechanism of transcriptional pausing. , 2006, Biochemical Society transactions.

[10]  Nick V Grishin,et al.  Structural basis for converting a general transcription factor into an operon-specific virulence regulator. , 2007, Molecular cell.

[11]  Ruchir Shah,et al.  RNA polymerase is poised for activation across the genome , 2007, Nature Genetics.

[12]  R. Young,et al.  A Chromatin Landmark and Transcription Initiation at Most Promoters in Human Cells , 2007, Cell.

[13]  Tahir H. Tahirov,et al.  Structural basis for transcription elongation by bacterial RNA polymerase , 2007, Nature.

[14]  M. O’Donnell,et al.  Replisome mechanics: insights into a twin DNA polymerase machine. , 2007, Trends in microbiology.

[15]  P. Babitzke,et al.  Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader , 2008, Proceedings of the National Academy of Sciences.

[16]  D. Price,et al.  Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay , 2008, Nucleic acids research.

[17]  Akira Hirata,et al.  The X-ray crystal structure of RNA polymerase from Archaea , 2008, Nature.

[18]  P. Cramer,et al.  Structure of an archaeal RNA polymerase. , 2008, Journal of molecular biology.

[19]  Jena Yamada,et al.  Core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation. , 2008, Structure.

[20]  Leemor Joshua-Tor,et al.  On helicases and other motor proteins. , 2008, Current opinion in structural biology.

[21]  Jens Michaelis,et al.  Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex , 2009, Nucleic acids research.

[22]  K. Murakami,et al.  Archaeal RNA polymerase. , 2009, Current opinion in structural biology.

[23]  P. Cramer,et al.  RNA polymerase II–TFIIB structure and mechanism of transcription initiation , 2009, Nature.

[24]  H. Handa,et al.  DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation. , 2009, Genes & development.

[25]  Paul Rösch,et al.  Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface. , 2010, The Biochemical journal.

[26]  R. Landick,et al.  Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. , 2009, Journal of molecular biology.

[27]  S. Block,et al.  E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase. , 2010, Journal of molecular biology.

[28]  A. Hoenger,et al.  Molecular basis of transcription initiation in Archaea , 2010, Transcription.

[29]  Andrew C. R. Martin,et al.  Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif , 2010, Nucleic acids research.

[30]  D. Fargo,et al.  Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II in Drosophila , 2010, Science.

[31]  D. Gilmour,et al.  Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex , 2010, Proceedings of the National Academy of Sciences.

[32]  Björn M. Burmann,et al.  A NusE:NusG Complex Links Transcription and Translation , 2010, Science.

[33]  I. Artsimovitch,et al.  Functional analysis of Thermus thermophilus transcription factor NusG , 2010, Nucleic acids research.